Adverse changes in small cerebral blood vessels due to type 2 diabetes (T2D) lead to cognitive impairment, memory deficits and dementias, and potentiate brain injury due to cerebrovascular accidents. The mechanisms are not fully known but detrimental changes in mitochondrial in endothelium appear to play a pivotal, initiating role. We have generated pilot data and developed new models to study the cerebral microcirculation during T2D and strokes. Our studies are conceptually innovative based on discoveries by our laboratory: (1) major sex-differences in mitochondrial abundance under normal conditions, (2) preferential effects on mitochondria in microvessels compared with arteries in T2D, (3) differential expression of mitodestructive and mitoprotective proteins in male and female blood vessels, (4) sex-dependent responses of mitochondria in the cerebral vasculature following strokes, and (5) major changes in vascular mitochondrial characteristics at sites distant from brain injury. Our studies are technically innovative based on new approaches to study the cerebral microcirculation of the mouse. First, we have developed a mouse model that genetically labels mitochondria only in endothelium with Dendra2 green/red photoswitchable fluorescent protein. Mitochondrial density, locations in endothelium, vascular diameters, and numbers (Rhodamine red) in the cerebral microcirculation can be simultaneously measured, at the same sites in multiple brain areas, for up to 12 months with multiphoton microscopy in mice anesthetized for each determination. Second, we have developed a high throughput method, which allows for the first time the determination of mitochondrial respiration in freshly harvested brain microvessel preparations from the mouse. We will extend this method to compare ATP production in the same sample by OXPHOS and glycolysis or the use of alternative fuels by mitochondria. Third, we will use RNAseq and Proteomics to elucidate mechanisms underlying changes observed during aging and T2D. These approaches are providing novel information on signaling pathways. We also will examine effectiveness of mitochondria-directed therapies in limiting damage and/or improving recovery to the microcirculation in T2D and strokes. Our overall hypothesis is that mitochondria in endothelium represent novel targets for sex-specific and disease-specific therapies. We have 2 aims. Aim 1: Characterize mitochondrial dynamics and vascular architecture of male and female mice under baseline conditions and during the development of T2D. We will: a) determine mitochondrial and vascular characteristics using in vivo multiphoton imaging in mice on a low or high fat diet, b) investigate mitochondrial and vascular changes in harvested microvessels during progression of T2D, c) elucidate mechanisms affecting mitochondrial and vascular dynamics during T2D, and d) explore treatment modalities. Aim 2: Investigate mitochondrial dynamics and vasculature architecture of male and female diabetic mice following transient ischemia. We will: a) determine mitochondrial and vascular changes using in vivo and ex vivo approaches in diabetic mice following transient middle cerebral artery occlusion (tMCAO)ischemic stress, b) elucidate mechanisms involved in changes in mitochondrial and vascular dynamics, and c) explore therapeutic approaches to improve mitochondrial and vascular function after ischemia in T2D mice.